mmg_233_2013_genetics_genomicswikiaorg-20200214-history
Endosymbiotic Gene Transfer: Organelle genomes forge eukaryotic chromosomes
Scientists discovered that mitochondrial and chloroplast DNA are present in the nuclear genome of their eukaryotic host over 20 years ago. The details of this occurence, however, are still being worked out (1). Timmis et al. provide a review of nuclear, organellar, and cyanobacterial and a-proteobacterial genomes in order to discuss clues of how the precursors to chloroplasts and mitochondria (respectively) were incorporated and now share their genomes with eukaryotic species (1). Evidence for incorporation of genetic material from the mitochondria and chloroplast into the nucleus comes from the fact that precursor cyanobacteria and proteobacteria encode 8,300 and 6,700 proteins while mitochondria encode 3-67 and chloroplasts 20-200 proteins (1). Endosymbiotic Gene Transfer Further evidence for gene transfer was elucidated in the 1980's when mitochondrial DNA was found in bacteria, yeast, and even chloroplast DNA in plant mitochondria. This lead to the term "promiscuous DNA" coined by John Ellis in 1982. Figure 1 illustrates the idea of promiscuous DNA in that genes from each bacterial ancestor for mitochondria and chloroplast transferred most of their DNA to the nucleus. The organelles are then dependent on the nucleus for production of proteins for the organelle to function. Additionally, chloroplast DNA has been found in mitochondria and the nucleus, but mitochondrial DNA has not been found in the chloroplast (1). Given the known information that gene transfer has occurred and is occuring still today, scientists wanted to develop techniques to study the rate of gene transfer from the organelle to the nucleus (1). Measuring Gene Transfer and Questions about the Mechanism Studying organelle to nucleus gene transfer became possible with the use of several molecular biology tools. Measuring mitochondrial --> nuclear genome transfer was performed in yeast, where it was found that transfer occurs at 2x10^-5 per cell per generation (1). In an elegant study by Huang et al, the rate of chloroplast DNA to the nuclear genome was elucidated. Figure 2 illustrates the process by which double selection markers were inserted into the chloroplast and selected for. One of the genes, neoSTLS2, ''was only designed to express when translocated to the nucleus due to its nuclear-specific promoter. Thus plants with the chloroplast selection marker ''aadA ''and the nuclear expression of ''neoSTLS2 were selected. The rate of gene transfer was determined to be 1 in 16,000. The next section describes this experiment (1). Additional questions about incorporation of organelle DNA into the nucleus include: #How is it transferred? #Where does the DNA integrate? #Why are not all genes transferred? While the answers to the questions are still being determined, it is hypothesized that bulk DNA or cDNA is how organelle DNA is transferred. In the bulk DNA view, DNA that escapes from the organelle is recombined with the nuclear DNA. The cDNA hypthesis is newer and says that strands of cDNA that have been edited and spliced are the transfer material (1). DNA does not integrate in a given spot in the nuclear genome. In fact, of the 16 chloroplast to nuclear translocations found by Huang et al, all were in different locations in the genome (see Ref. 2). Gene transfer of all genes from the organelle would be detrimental to the organelle and cell due to the potential for redox toxicity and the fact that proteins needed for electron transport should be encoded in the organelle itself (1). Thus, movement of some genes to the nucleus allow the organelle to control only what is needed, but in turn makes the organelle completely dependent on the host nuclear DNA. In this way, organelle DNA helped to create the eukaryotic chromosomes we study today. Transfer of neoSTLS2 from the chloroplast into the nucleus In their paper, "Direct measurement of the transfer rate of chloroplast DNA into the nucleus ," Huang et al measured the transfer rate of chloroplast DNA into the nucleus of tobacco plants via an engineered neomycin resistant phosphotransferase gene (neoSTLS2). The authors mention that more is known about mitochondrial gene transfer, but little is known about the rate of chloroplast gene transfer into the nucleus. The neoSTLS2 gene is specific for the nucleus. Therefore insertion of it into the the chloroplast genome would enable the authors to quantify translocation to the nucleus. The plasmid constructed for insertion into the chloroplast genome (plastome) is indicated in Figure 3. In brief, the neoSTLS2 was ligated into the PRV111A vector, and transformed with a plastid-selectable aminoglycoside 3'-adenyltransferase marker gene (aadA) into plastids of the tobacco plant via biolistic bombardment (gene gun) and plants with both plasmids grown. Seedlings were selected for kanamycin (neoSTLS2) and spectinomycin (aadA) resistance. The neoSTS2 gene was designed to only be expressed when transposed to the nucleus, allowing the plant to be selected for kanamycin resistance. This was enabled by inserting a potato nuclear intron, ST-LS1 within the reading frame of the neoSTLS2 gene within the chloroplast, rendering the protein non-functional. Thus if localization to the nucleus has occured, the gene is functional and can be selected for. Upon screening seedlings, 18 kanamycin resistant plants (of 250,000) were found, indicating translocation to the nucleus (2 of which were not the STLS2 gene, leaving 16 insertions). These same plants were also spectinomycin sensitive indicating that the aadA gene was not translocated to the nucleus and that chloroplast transfer from the parent chloroplast did not occur. The final data from the authors show that chloroplast DNA is transferred at a rate of 1 in 16,000 tobacco pollen grains. In a caveat to this data, the authors did not measure deletion of chloroplast genes from the nucleus, but do mention that insertion and deletion are thought to be balanced evolutionarily. References 1. Timmis et al. "Endosymbiotic Gene Transfer: Organelle genomes forge eukaryotic chromosomes." Nature Reviews.(2004). Volume 5 (2), pages 123-35 PMID: 14735123 2. Huang CY, Ayliffe MA, and Timmis JN. "Direct measurement of the transfer rate of chloroplast DNA into the nucleus." Nature. (2003). 422(6927): 72-6 PMID: 12594458 3. University of Washington, Center for Comparative Genomics. "Biolistic Bombardment, Cell or Tissue Transformation System." http://depts.washington.edu/genomelb/BiolisticBombardment&MiscV3.html 4. Wikipedia. "Nicotiana tabacum." http://en.wikipedia.org/wiki/Nicotiana_tabacum